Ion channels reside in a cell membrane to control crossing of ions into the cell. Specifically, ion channels have a structure spanning the cell membrane, and open and close on various stimulations to bring ions into and out of the cell. The ion channels therefore constitute one class of molecules that elicit important biological reactions such as muscle contraction, release of neurotransmitters, and secretion of insulin. For example, the electrical signal transmission in nerve cells requires sodium channels or potassium channels. The potassium channels are essential for muscle contraction. Some of the different forms of ion channels are potential-dependent ion channels, which open and close according to a potential difference across the cell membrane, and receptor-activated ion channels, which open and close in response to binding of a ligand forming a complex with the receptor.
The inventors of the present invention have analyzed ion channel-mediated electrical activity using Ascidiacea, which is a member of Protochordata.
Ascidiacea belongs to Urochordata, and is a member of Chordata as are cephalochordates (for example, lancelets) and vertebrates (for example, frogs, humans). Unlike vertebrates, the chordate Ascidiacea does not have any redundant genes, preserving the basic form of chordates before gene redundancy. The tadpole larva of Ascidiacea has the simplest (original) form of organization among the chordates. This makes the chordate Ascidiacea the most suitable model animal for elucidation of development mechanism of vertebrates in general including humans. It is therefore anticipated that genes expressed in Ascidiacea will be applicable to medicine such as gene therapy and regenerative medicine, and various other fields including environment and food.
The inventors have previously reported a gene expression profile in the tailbud embryos of Ciona intestinalis (see Non-Patent Publication 1, for example).
Further, the inventors have performed EST (expressed sequence tag) analysis for 76,920 genes on the 3′ sequence and 76,250 genes on the 5′ sequence, concerning mRNA of genes expressed in a fertilized egg, cleaving embryos, tailbud embryos, larva, juvenile, or sperm of Ciona intestinalis. Expression of 5,000 genes selected from these genes was then examined by in situ hybridization. It was found as a result that about 500 genes were specifically expressed in the tailbud embryos, larva, or tissues or organs of juvenile. Among these genes, the entire base sequences of the mRNA (cDNA) of 261 genes were determined. (See Patent Publication 1, for example.)
Further, the inventors have performed EST analysis for about 23,000 genes on the 3′ sequence and about 23,000 genes on the 5′ sequence, concerning mRNA of genes expressed in the tailbud embryos and larva of Ciona intestinalis. Expression of about 3,000 genes selected from genes was examined by in situ hybridization. It was found as a result that about 200 genes were expressed in the nervous system (central nervous system or peripheral nervous system) of Ascidiacea. Among these genes, the entire base sequences of mRNA (cDNA) of 108 genes were determined. (See Patent Publication 2, for example.)
[Patent Publication 1]
Japanese Laid-Open Patent Publication No. 2004-57129 (published on Feb. 26, 2004)
[Patent Publication 2]
Japanese Laid-Open Patent Publication No. 2004-57127 (published on Feb. 26, 2004)
[Non-Patent Publication 1]
Development 128, 2893-2904 (2001)
In ion channel research, there has been established an experimental system in which ion channels, expressed for example in the oocyte of Xenopus laevis by a gene introducing technique are analyzed by techniques in electrophysiology. Such an experimental system is used to examine functions and control mechanism of ion channels, or influence of drugs on ion channel activity.
In order to examine influence of drugs on ion channels, it is generally required to capture a minute current that is generated by the movement of ions in and out of the cell. Measurement of cell membrane voltage from the cell conventionally requires a direct electrical measurement or a quantitative analysis using a voltage-sensitive pigment. However, it has been difficult with these techniques to obtain information concerning local membrane potential of the cell. Particularly, analysis has been impossible on materials of a small structure, such as sperm, to which the electrophysiological techniques are difficult to apply. Further, the experiment requires a skilled technique and is laborious.
Further, the screening techniques used in the research of enzymes and receptors are not directly applicable to ion channel research. This has slowed the development of drugs that involve ion channels. In fact, the only drugs that are known to act on ion channels are, for example, calcium antagonists available as hypotensive drugs, and local anesthetics.
Despite a wide range of studies that have been made on relations between physiological activity of organisms and ion channel molecules. Not all channel-like proteins have been identified and many questions remain unanswered. It is therefore useful to comprehensively analyze channel functions in simple organisms, from the genomic level to organism level.